1. Field of the Invention
The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.
2. Background Information
Fatty acids are organic acids which are present in lipids, and vary in carbon content from C.sub.2 to C.sub.34. In biosynthetic reactions, they are often covalently bound through a thioester linkage to Coenzyme A (CoA) to form acyl-CoA or to acyl carrier proteins (ACPs) to form acyl-ACP. Fatty acids serve numerous physiological functions. For example, they may be joined through an ester linkage to fatty alcohols to form waxes, and to glycerol molecules through ester linkages to form triglycerides.
Linear wax esters are lipophilic compounds containing a long chain fatty alcohol esterified to a long chain fatty acid. In naturally occurring wax esters, the fatty alcohol and fatty acid moieties typically each contain 16 to 24 carbons and may contain one or more unsaturations. Such wax esters are found in a number of different organisms. For instance, wax esters are the principal component of spermaceti oil which, until recently, was obtained from the head cavity of sperm whales (Physeter macrocephalus). In 1979 the United states banned the import of all cetacean products and in 1982 the European Community enacted similar legislation. Since this time, the only natural source of wax esters on a commercial scale has been the seeds of jojoba (Simmondsia chinensis Link), a bush or shrub that is adapted to growth in hot arid habitats. In jojoba, waxes are stored in the seeds of the plant where they serve as a means of energy storage for developing seedlings. Wax esters have also been found in several species of bacteria such as Acinetobacter calcoaceticus, a gram negative aerobic bacteria that accumulates wax esters when grown under nitrogen limited conditions (Fixter et al.1986). Although these organisms are very diverse, examination of the chemical structures of the waxes they produce reveals that these waxes are very similar to each other structurally, in terms of their fatty acid and fatty alcohol components, carbon chain lengths and degree of saturation.
Wax esters have important commercial applications in a variety of technical areas, including the medical, cosmetics and food industries as well as their more traditional usage as lubricants for mechanical parts and the like. Consequently, enzymes and enzymatic pathways which are involved in production of waxes have great potential utility for more efficient production of known waxes, as well as for production of useful novel compounds.
Likewise, fatty alcohols and aldehydes have important commercial uses, e.g. as solvents, lubrication oil additives, plasticizers, nonionic surfactants, in pharmaceutical compositions as salves and lotions and as ingredients in cosmetics.
The wax esters obtained from jojoba can replace sperm whale oil in most or all traditional uses. They are useful for applications in cosmetics, as a lubricant, as an additive for leather processing, as a carrier for pharmaceuticals and as a solvent. Hydrogenation of the wax to eliminate double bonds produces a hard wax which is useful for surface treatments, in textile sizing, in coating paper containers and in cosmetics (eg., lipstick and creams). Sulphurization of the wax or other modifications make the substance useful in specialty lubricant applications, as a textile softener, as a component of printing inks, and as a component in many technical products such as corrosion inhibitors, surfactants, detergents, disinfectants, plasticizers, resins and emulsifiers. For some of these applications the fatty alcohol derived by hydrolysis of the wax ester is the most valuable ingredient derived from the wax ester. However, because the yield of the jojoba plant is relatively low, the oil is relatively expensive compared with edible oils from plants or technically comparable materials from petroleum. Thus, there has been interest in developing an alternate biological source of wax esters and long-chain alcohols. One possibility, in this respect, is to modify a microbial species for efficient production of wax esters or long-chain alcohols. Another possibility is to transfer the capability to produce wax esters or long-chain fatty alcohols to highly productive plant species.
Acyl-CoA compounds are utilized as substrates by fatty acyl reductases to form primary fatty alcohols in a two step process in which aldehydes are intermediate products. The alcohol product can then be joined through an ester linkage to a fatty acid to form a wax. Thus, fatty acyl reductases are involved in the production of fatty alcohols, aldehydes and wax esters.
The most detailed published information concerning wax ester biosynthesis concerns wax biosynthesis in jojoba where it appears that two enzymes catalyze the formation of wax esters (Pollard and Metz 1995; Metz et al. 1995). The first step of the pathway is catalyzed by a fatty acyl-CoA reductase which is highly substrate-specific for tetracosenoyl-CoA (a 24 carbon acyl-CoA), and is known to catalyze the formation of a long chain alcohol directly from this substrate via an aldehyde intermediate (Pollard and Metz 1995). The second enzyme,an acyl-CoA-fatty alcohol transferase catalyzes the formation of an ester linkage between acyl-CoA and a fatty alcohol to yield a wax ester. Assays on this enzyme, found it to be acyl-CoA specific, preferring C.sub.20 -monounsaturated acyl-CoA's and C.sub.14 and C.sub.18 mono- and di-unsaturated fatty alcohols (Metz et al. 1995).
Relatively little is known in detail about the biochemical mechanisms of wax ester production in bacteria. It is generally believed that the starting substrate is either acyl-ACP or acyl-CoA. The acyl compound is thought to be reduced to the corresponding aldehyde by an acyl-ACP or acyl-CoA reductase. An aldehyde intermediate has been proposed to occur based on the observation of a constitutive NAD-dependent long chain alkanal dehydrogenase and an inducible (induced in the presence of alkanes) NADP-dependent alkanal dehydrogenase in crude extracts of Acinetobacter strain HO1-N (Fox et al. 1992; Singer and Finnerty 1985c). With this observed activity from these two enzymes, it was proposed that one, or both, of the enzymes might be catalyzing the reverse reaction, reducing acyl-ACP to the corresponding aldehyde. However, no direct evidence was put forth to support this idea. The second step in wax ester formation involves the reduction of the fatty aldehyde to its corresponding fatty alcohol. Here again, the same logic was proposed. Two independent reports describe cofactor dependent and independent fatty alcohol dehydrogenases which have been proposed to play a role in wax ester biosynthesis (Fox et al. 1992; Singer and Finnerty 1985b). However, these reports are based on the use of crude extracts which may contain many different enzymes of similar function. Thus, nothing about the relevant enzymes is known with any degree of certainty based on previous published studies.
Several U.S. patents disclose fatty acyl reductase proteins isolated from plants.
U.S. Pat. No. 5,403,918 describes a partially purified fatty acyl reductase protein produced from jojoba embryos with activity towards acyl substrates having chain lengths from 16 to 24 carbon atoms. This enzyme is NADPH dependent, has a molecular mass of about 53 kD, and prefers very long chain acyl-CoA substrates.
U.S. Pat. No. 5,411,879 discloses partially purified proteins of about 32 kD and 47 kD obtained from jojoba embryos which are proposed to be components of an NADPH-dependent fatty acyl-CoA reductase, and two short amino acid sequences obtained from the 47 kD protein.
U.S. Pat. No. 5,370,996 discloses the nucleic acid sequence and translated amino acid sequence of a jojoba fatty acyl reductase.
From the limited amount of information available about the fatty acyl reductases from jojoba it appears that the enzyme has a strong substrate preference for acyl groups of 20 carbons or more in length. By contrast, the fatty acyl reductase from A. calcoaceticus appears to preferentially utilize shorter chain fatty acyl substrates. Growth of cultures in minimal mineral media with succinate or acetate as a carbon source produced wax esters in which about 50% were composed of two 16 carbon acyl groups and another 40% had one 16 carbon acyl group (Dewitt et al. 1982). By growing the bacteria in minimal mineral media with hexadecane (a 16 carbon alkane) as a carbon source 100% of the waxes were 32 carbons in length. Incubation of cultures in longer chain alkanes was found to give rise to wax ester compositions of C.sub.2n, C.sub.2n-2 and C.sub.2n-4 (Dewitt et al. 1982). Thus, the availability of genes encoding the bacterial enzyme may permit the production of fatty alcohols and wax esters of different chemical composition than the jojoba enzyme. Also, since it is not known what regulates the activity of fatty acyl reductases, the bacterial enzymes described here may have different mechanisms of regulation than the jojoba enzyme. It is also shown that the enzymes of this invention are closely related to an enzyme that participates in the formation of mycolic acid, a lipophilic constituent of the human pathogen Mycobacterium tuberculosis. Thus, the detailed information concerning the genes and enzymes of this invention may be used to facilitate the design of new drugs that inhibit the growth of this species by inhibiting the acyl-CoA reductase of this invention. The enzyme of the present invention shows significant amino acid sequence similarity to two open reading frames from otherwise anonymous cDNA clones from the plant Arabidopsis thaliana (L.) and describe methods for showing the acyl CoA reductase activity of the gene products corresponding to those plant genes. The Arabidopsis genes can be used to obtain structurally similar genes from other plant species by a variety of methods that are known to those skilled in the art. In particular, the Arabidopsis genes may be used as hybridization probes to screen cDNA or genomic libraries prepared from other species. Alternately, the genes from other species may be recognized by scanning databases of partially or completely sequenced cDNA clones for clones that exhibit significant nucleotide or deduced amino acid sequence similarity. For instance genes that exhibit greater than about 60% overall nucleotide similarity and as little as about 30% deduced amino acid sequence similarity are typically considered to be genes or gene products of similar or identical function. Extensive collections of such partial sequences are already available for a number of plants species and the collections are expected to expand in the near future. In addition, the sequences of the Arabidopsis gene products may be used to design degenerate oligonucleotide primers that encode all codons capable of producing a given region of amino acid sequence. Pairs of primers can then be used to amplify a fragment of a corresponding gene from genomic DNA or cDNA of another species, and the amplified fragment may then be used as a hybridization probe form the complete gene or cDNA sequence.